APETALA3-nuclease hybrid protein: a potential tool for APETALA3 target gene mutagenesis

Abstract

Direct target genes of homeotic genes are largely unknown in plants. The class B homeotic gene APETALA3 ( AP3) is required for petal and stamen identities. The AP3 gene encodes a MADS-domain containing protein which forms heterodimers in vitro with the second class B homeotic protein PISTILLATA ( PI). Here, we describe a new strategy that can be used to isolate mutants of genes that are immediate targets of AP3 or AP3/PI. The strategy is based on providing a nuclease activity to AP3 by translationally fusing the F N nuclease domain of the FokI restriction enzyme. In electro-mobility shift assays, AP3-F N/PI heterodimers display the same binding specificity for CArG-box elements as AP3/PI heterodimers, although with a lower affinity. Transgenic lines carrying the AP3-F N fusion gene under control of the 35S promoter were obtained. The 35S:: AP3-F N construct is able to partially suppress the ap3-1 mutant phenotype showing that the AP3 part of the hybrid protein is functional in vivo. When crossed with the DNA-break repair deficient mutant uvh1, offspring were obtained that showed, to various degrees, a lack of fertility consistent with the role of AP3 in gamete development. The mutant phenotypes are inherited to the next generation. This is the first report of a strategy designed to create mutants of genes directly regulated by a homeotic gene.